The gram-negative bacterium Pseudomonas aeruginosa is a widespread bacterium that is pathogenic for humans and that constitutes a major health risk especially to neonates and to people having weakened resistance. Besides its major clinical significance, the antibiotic resistances that are frequently present and the formation of toxins, especially the highly toxic exotoxin A (Woods, D. E. and Iglewski, B. H., Rev. Infect. Dis. 5, 714-722 (1983), Pseudomonas aeruginosa is one of the most important bacterial causes of cases of food poisoning. Conventional processes require at least 4 days for the detection of Pseudomonas aeruginosa. There is therefore an urgent need for the development of rapid processes for detecting Pseudomonas aeruginosa in food and in clinical samples.
In recent years, a number of new methods have been developed for routine use in detecting particular microorganisms. These include immunological processes based on the use of polyvalent or monoclonal antibodies and processes in which nucleic acid probes are used for detection by means of hybridisation to organism-specific nucleic acids. Further methods that have been described are those processes which are based on a specific nucleic acid amplification, with or without a subsequent confirmation reaction by nucleic acid hybridisation. Processes used for the amplification of nucleic acids are, for example, the polymerase chain reaction (PCR) [U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188], the ligase chain reaction [WO Publication 89/09835], “self-sustained sequence replication” [EP 329,822], the “transcription based amplification system” [EP 310,229] and the Qβ RNA-replicase system [U.S. Pat. No. 4,957,858].
The mentioned nucleic-acid-based processes are so sensitive that, in contrast to conventional microbiological processes, it is possible to dispense with, or considerably curtail, a lengthy increase in quantity of the microorganism being detected from the sample under investigation. Testing for the presence or absence of the microorganism in question is therefore generally concluded within one day when using the mentioned nucleic-acid-based processes, thereby achieving a considerable reduction in time, especially when conventional processes require several days or weeks for detection.
Various PCR-based processes for the detection of Pseudomonas aeruginosa have been described. By amplifying a region of DNA having a length of 369 bp from the exotoxin A gene it has been possible to detect the presence of strains of the species Pseudomonas aeruginosa selectively [Khan et al. (1994), Appl. Environ. Microbiol. 60, 3739-3745]. Even though no bacteria of other species were detected using that PCR system, an amplified product was observed in only 96% of the 130 Pseudomonas aeruginosa strains tested in total. Consequently, that PCR system is of only limited suitability for establishing a rapid process by means of which the presence of all strains of Pseudomonas aeruginosa can be detected reliably.
With the aid of a further, recently published process based on a multiplex PCR it has been possible to detect, selectively, fluorescent pseudomonads on the one hand and Pseudomonas aeruginosa on the other hand [De Vos et al. (1997), J. Clin. Microbiol. 35, 1295-1299]. Using that process, it was possible to detect each of the 150 isolates of Pseudomonas aeruginosa tested in total. It is, however, disadvantageous that the oprL gene used for the selective detection of Pseudomonas aeruginosa is also highly conserved in other species of the Pseudomonas genus. Thus, the amino acids that are coded for in the region of the binding sites of the primers used by Voss et al. are identical in Pseudomonas putida and Pseudomonas aeruginosa. The detection of Pseudomonas aeruginosa is accordingly based merely on a few different base pairs caused by the variation in the third position of particular amino acid codons, which on the basis of experience carries a high risk of false-positive and/or false-negative results occurring.
In addition, because of the high degree of conservation of the oprI and oprL genes, the multiplex PCR system described is unlikely to offer a possible means of detecting—for example by the use of various probes subsequently to the PCR reaction—other clinically significant species of the Pseudomonas genus, such as, for example, Pseudomonas fluorescens, Pseudomonas mendocina, Pseudomonas putida or Pseudomonas stutzeri. 
An aim of the invention described herein was to establish nucleic acid sequences whose use as primers and/or probes would ensure detection, in as complete a manner as possible, of all representatives of the species Pseudomonas aeruginosa. A further aim of the invention was to identify a region of the genome having sufficiently high sequence variability within different species of the Pseudomonas genus to allow, optionally, the detection of other species of the Pseudomonas genus as well, for example by using different variants of primers and/or probes in the PCR or subsequently to the PCR.
Depending on the size of the group of microorganisms to be detected and the evolutionary relatedness (similarity) of microorganisms to be excluded (that are not to be detected), detection based on differential DNA sequences requires very extensive preliminary work in order to identify suitable DNA sequences that have the desired specificity in the particular case. The invention described herein relates to such DNA sequences, by means of which the rapid detection of bacteria of the Pseudomonas genus, especially of Pseudomonas aeruginosa, is possible.
Also known are nucleic acid molecules that can be used as probes or primers for the detection of microoganisms, especially for the detection of Pseudomonas aeruginosa (WO 96/00298 and Acta Microbiologica Polonica, 44 (1995) 111-117) the nucleic acid molecules being obtained from the 16S-23S intergenic region. Accordingly, it is possible for Pseudomonas aeruginosa to be distinguished from other Pseudomonas species and also from bacteria of other genera.
It is also known that, for bacteria that do not belong to Pseudomonas species, the 23S-5S intergenic region can be successfully used for isolating species- and genera-specific nucleic acid molecules (J. Applied Bakteriology, 80 (1996) 244-251 and EP 0 739 988).